Limitations of the fluorescent probe viability assay.

Molecular toxicology Pub Date : 1989-10-01
E J Massaro, K H Elstein, R M Zucker, K W Bair
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引用次数: 0

Abstract

In vitro assessment of the efficacy/capacity of toxicants (e.g., cancer chemotherapeutic agents, environmental pollutants, etc.) to damage/kill cells and/or inhibit growth (cell duplication) requires accurate measurement of target cell viability as a function of exposure. Rapid measurement of viability, such as can be achieved employing fluorescent probes of metabolic function in combination with instrumental analysis, is highly desirable. However, we observe that exposure to chemicals (of unrelated type) complicates the interpretation of viability data and, in the case of perturbed cells, questions the validity of viability growth assays based on intrinsic enzyme activity. Viability commonly is determined flow cytometrically (FCM) by the carboxyfluorescein diacetate (CFDA)/propidium iodide (PI) assay. Nonfluorescent CFDA is taken up by diffusion and converted via cytoplasmic esterase-catalyzed hydrolysis to carboxyfluorescein (CF), a negatively charged fluorescent molecule that is retained (incompletely) by the cell. As such, if CF fluorescence intensity is a relative measure of enzyme activity, it also can be considered an index of cellular vigor (metabolic rate). It is generally accepted that the viable cell excludes both basic dyes, such as PI, and acidic dyes, such as trypan blue, and uptake is indicative of irreversible cellular injury presaging cell death. We observe that, following incubation for 4 h with 0.5-1.0 microM tributyltin (TBT), a potent environmental toxicant, murine erythroleukemic cells (MELC) exhibit enhanced (supranormal) CF fluorescence compared to control cells. Apparent cell volume (ACV) is unaltered, and because such cells exclude PI, they are considered viable in terms of the CFDA/PI assay. However, rate of growth (increase in cell number over 48 h) is depressed, suggesting that supranormal CF fluorescence, even in the absence of PI uptake, is indicative of cellular perturbation. In effect, although CF fluorescence is the product of an enzyme-catalyzed reaction and, therefore, an indicator of vital function (enzyme activity), it apparently is not a reliable index of cellular vigor. At higher TBT concentrations (greater than 1.0, but less than 50.0 microM), the cells exhibit both increased CF fluorescence and PI fluorescence and are growth inhibited. MELC exposed to the cancer chemotherapeutic agents adriamycin, m-AMSA, or crisnatol (Burroughs Wellcome 770U82) also exhibit increased cellular CF fluorescence. However, rate of growth is decreased and ACV increased. The latter, measured either as a function of electrical resistance (Coulter volume) or by the FCM parameter axial light loss could account for the increase in mean CF fluorescence.(ABSTRACT TRUNCATED AT 400 WORDS)

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荧光探针活力测定的局限性。
在体外评估有毒物质(如癌症化疗药物、环境污染物等)损害/杀死细胞和/或抑制生长(细胞复制)的功效/能力,需要准确测量靶细胞活力作为暴露的函数。非常需要快速测量活力,例如可以使用代谢功能的荧光探针与仪器分析相结合来实现。然而,我们观察到,暴露于化学物质(不相关类型)使活力数据的解释复杂化,并且在受干扰细胞的情况下,质疑基于内在酶活性的活力生长测定的有效性。生存能力通常是通过二乙酸羧基荧光素(CFDA)/碘化丙啶(PI)测定的流式细胞术(FCM)来确定的。非荧光CFDA被扩散吸收,并通过胞质酯酶催化水解转化为羧基荧光素(CF),这是一种带负电的荧光分子,被细胞保留(不完全保留)。因此,如果CF荧光强度是酶活性的相对度量,它也可以被认为是细胞活力(代谢率)的指标。人们普遍认为,活细胞不包括碱性染料,如PI和酸性染料,如台盼蓝,摄取是不可逆的细胞损伤预示着细胞死亡。我们观察到,与0.5-1.0微米的三丁基锡(TBT)(一种强效环境毒物)孵育4小时后,小鼠红细胞白血病细胞(MELC)与对照细胞相比表现出增强的(异常的)CF荧光。表观细胞体积(ACV)不变,由于这些细胞排除了PI,因此根据CFDA/PI检测,它们被认为是活的。然而,生长速度(超过48小时的细胞数量增加)下降,表明即使在没有PI摄取的情况下,异常的CF荧光也表明细胞受到扰动。实际上,虽然CF荧光是酶催化反应的产物,因此是一种重要功能(酶活性)的指标,但它显然不是细胞活力的可靠指标。在较高的TBT浓度(大于1.0,但小于50.0微米)下,细胞的CF荧光和PI荧光均增加,生长受到抑制。暴露于癌症化疗药物阿霉素、m-AMSA或crisnatol (Burroughs Wellcome 770U82)的MELC也表现出细胞CF荧光增加。但是,生长速率降低,ACV增加。后者,作为电阻(库尔特体积)的函数或通过FCM参数测量,轴向光损失可以解释平均CF荧光的增加。(摘要删节为400字)
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Xenobiotic regulation of glutathione S-transferase Ya gene expression. Review: gene amplification--a cellular response to genotoxic stress. Limitations of the fluorescent probe viability assay. Induction of a novel damage-specific DNA binding protein correlates with enhanced DNA repair in primate cells. Induction of a novel damage-specific DNA binding protein correlates with enhanced DNA repair in primate cells.
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